Sevoflurane Inhibits Glutamate-Aspartate Transporter and Glial Fibrillary Acidic Protein Expression in Hippocampal Astrocytes of Neonatal Rats Through the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) Pathway.

BACKGROUND: The mechanisms underlying general anesthesia-induced neurotoxicity are unclear. Astrocytes have been recognized as important contributors to neuronal development. Until now, the response of the astrocytes to neonatal general anesthetic exposure has been unreported. METHODS: Postnatal day 7 rats received 2.5% sevoflurane for 6 hours. Expressions of glial fibrillary acidic protein (GFAP) and glutamate-aspartate transporter (GLAST) and phosphorylation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway were detected on days 1, 3, 7, and 14 after sevoflurane inhalation. In addition, cultured astrocytes were exposed to 2.5% sevoflurane for 2 hours and GFAP, GLAST expressions, and JAK/STAT phosphorylation were evaluated. Furthermore, we pharmacologically disrupted JAK/STAT signaling in vivo by treatment with the JAK/STAT inhibitor AG490 and in vitro by treatment with JAK inhibitor I to detect the consequent expression of GFAP and GLAST. RESULTS: Sevoflurane induced a robust decrease of GFAP and GLAST expression in hippocampal tissue compared with sham control groups at 1 to 14 days after sevoflurane exposure. Immunohistochemistry showed colocalization of GFAP, GLAST, and pSTAT3 in the hippocampal CA1 region. Western blot analysis also revealed a significant decrease of pJAK1, pJAK2, and pSTAT3 in the sevoflurane group. In vitro study showed that GFAP, GLAST, pJAK1, pJAK2, and pSTAT3 expressions in cultured astrocytes were remarkably decreased at 24 to 48 hours after sevoflurane treatment. Either AG490 or JAK inhibitor I significantly decreased expressions of GFAP and GLAST in hippocampus or cultured astrocytes. CONCLUSIONS: Astrocytic GLAST was inhibited by sevoflurane in the hippocampus of neonatal rats. Inactivation of the JAK/STAT pathway possibly contributes to this effect of sevoflurane. Astrocytic dysfunction induced by sevoflurane may contribute to its neurotoxicity in the developing brain.

Inhibition of microglial activation contributes to propofol-induced protection against post-cardiac arrest brain injury in rats.

It has been suggested that propofol can modulate microglial activity and hence may have potential roles against neuroinflammation following brain ischemic insult. However, whether and how propofol can inhibit post-cardiac arrest brain injury via inhibition of microglia activation remains unclear. A rat model of asphyxia cardiac arrest (CA) was created followed by cardiopulmonary resuscitation. CA induced marked microglial activation in the hippocampal CA1 region, revealed by increased OX42 and P2 class of purinoceptor 7 (P2X7R) expression, as well as p38 MAPK phosphorylation. Morris water maze showed that learning and memory deficits following CA could be inhibited or alleviated by pre-treatment with the microglial inhibitor minocycline or propofol. Microglial activation was significantly suppressed likely via the P2X7R/p-p38 pathway by propofol. Moreover, hippocampal neuronal injuries after CA were remarkably attenuated by propofol. In vitro experiment showed that propofol pre-treatment inhibited ATP-induced microglial activation and release of tumor necrosis factor-α and interleukin-1ß. In addition, propofol protected neurons from injury when co-culturing with ATP-treated microglia. Our data suggest that propofol pre-treatment inhibits CA-induced microglial activation and neuronal injury in the hippocampus and ultimately improves cognitive function. We proposed a possible mechanism of propofol-mediated brain protection after cardiac arrest (CA). CA induces P2X7R upregulation and p38 phosphorylation in microglia, which induces release of TNF-α and IL-1ß and consequent neuronal injury. Propofol could inhibit microglial activation and alleviate neuronal damage. Our results suggest propofol-induced anti-inflammatory treatment as a plausible strategy for therapeutic intervention in post-CA brain injury.

Activation of the Mammalian Target of Rapamycin in the Rostral Ventromedial Medulla Contributes to the Maintenance of Nerve Injury-Induced Neuropathic Pain in Rat.

The mammalian target of rapamycin (mTOR), a serine-threonine protein kinase, integrates extracellular signals, thereby modulating several physiological and pathological processes, including pain. Previous studies have suggested that rapamycin (an mTOR inhibitor) can attenuate nociceptive behaviors in many pain models, most likely at the spinal cord level. However, the mechanisms of mTOR at the supraspinal level, particularly at the level of the rostral ventromedial medulla (RVM), remain unclear. Thus, the aim of this study was to elucidate the role of mTOR in the RVM, a key relay region for the descending pain control pathway, under neuropathic pain conditions. Phosphorylated mTOR was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the RVM after spared nerve injury- (SNI-) induced neuropathic pain. Moreover, in SNI rat brain slices, rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons. Finally, intra-RVM microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain. In conclusion, our data provide strong evidence for the role of mTOR in the RVM in nerve injury-induced neuropathic pain, indicating a novel mechanism of mTOR inhibitor-induced analgesia.

Genetic association of CHEK2, GSTP1, and ERCC1 with glioblastoma in the Han Chinese population.

Glioblastoma (GBM), a deadly brain tumor, is the most malignant glioma. It mainly occurs in adults and occurs significantly more in males than in females. We genotyped 19 tag single nucleotide polymorphisms (tSNPs) from 13 genes in a case-control study of the Han Chinese population to identify genetic factors contributing to the risk of GBM. These tSNPs were genotyped by Sequenom MassARRAY RS1000. Statistical analysis was performed using χ(2) test and SNPStats, a website software. Using χ(2) test, we found that the distribution of two tSNPs (rs2267130 in checkpoint kinase 2 (CHEK2), p = 0.040; rs1695 in GSTP1, p = 0.023) allelic frequencies had significant difference between cases and controls. When we analyzed all of the tSNPs using the SNPStats software, we found that rs1695 in GSTP1 decreased the risk of GBM in log-additive model (OR = 0.56, 95% CI, 0.34-0.94, p = 0.022). Besides, we found that there is an interaction between rs3212986 in excision repair cross-complementing group 1 (ERCC1) and gender under codominant and recessive models. The gene polymorphisms in CHEK2, GSTP1, and ERCC1 may be involved in GBM in the Han Chinese population. Since our sample size is small, further investigation needs to be performed.

Protective effect of quercetin against oxidative stress and brain edema in an experimental rat model of subarachnoid hemorrhage.

Quercetin has been demonstrated to play an important role in altering the progression of ischemic brain injuries and neurodegenerative diseases by protecting against oxidative stress. The effects of quercetin on brain damage after subarachnoid hemorrhage (SAH), however, have not been investigated. This study was designed to explore the effects of quercetin on oxidative stress and brain edema after experimental SAH using four equal groups (n = 16) of adult male Sprague-Dawley (SD) rats, including a sham group, an SAH + vehicle group, an SAH + quercetin10 group, and an SAH + quercetin50 group. The rat SAH model was induced by injection of 0.3 ml of non-heparinised arterial blood into the prechiasmatic cistern. In the SAH + quercetin10 and SAH + quercetin50 groups, doses of 10 mg/kg and 50 mg/kg quercetin, respectively, were directly administered by intraperitoneal injection at 30 min, 12 h, and 24 h after SAH induction. Cerebral tissue samples were extracted for enzymatic antioxidant determination, lipid peroxidation assay, caspase-3 activity and water content testing 48 h after SAH. Treatment with a high dose (50 mg/kg) of quercetin markedly enhanced the activities of copper/zinc superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GSH-Px), and treatment with this dose significantly reduced the level of malondialdehyde (MDA). Caspase-3 and brain edema was ameliorated and neurobehavioral deficits improved in rats that received the high dose of quercetin. The findings suggest that the early administration of optimal dose of quercetin may ameliorate brain damage and provide neuroprotection in the SAH model, potentially by enhancing the activity of endogenous antioxidant enzymes and inhibiting free radical generation.

Meshed neuronal mitochondrial networks empowered by AI-powered classifiers and immersive VR reconstruction.

Mitochondrial networks are defined as a continuous matrix lumen, but the morphological feature of neuronal mitochondrial networks is not clear due to the lack of suitable analysis techniques. The aim of the present study is to develop a framework to capture and analyze the neuronal mitochondrial networks by using 4-step process composed of 2D and 3D observation, primary and secondary virtual reality (VR) analysis, with the help of artificial intelligence (AI)-powered Aivia segmentation an classifiers. In order to fulfill this purpose, we first generated the PCs-Mito-GFP mice, in which green fluorescence protein (GFP) could be expressed on the outer mitochondrial membrane specifically on the cerebellar Purkinje cells (PCs), thus all mitochondria in the giant neuronal soma, complex dendritic arborization trees and long projection axons of Purkinje cells could be easily detected under a laser scanning confocal microscope. The 4-step process resolved the complicated neuronal mitochondrial networks into discrete neuronal mitochondrial meshes. Second, we measured the two parameters of the neuronal mitochondrial meshes, and the results showed that the surface area (µm2) of mitochondrial meshes was the biggest in dendritic trees (45.30 ± 53.21), the smallest in granular-like axons (3.99 ± 1.82), and moderate in soma (27.81 ± 22.22) and silk-like axons (17.50 ± 15.19). These values showed statistically different among different subcellular locations. The volume (µm3) of mitochondrial meshes was the biggest in dendritic trees (9.97 ± 12.34), the smallest in granular-like axons (0.43 ± 0.25), and moderate in soma (6.26 ± 6.46) and silk-like axons (3.52 ± 4.29). These values showed significantly different among different subcellular locations. Finally, we found both the surface area and the volume of mitochondrial meshes in dendritic trees and soma within the Purkinje cells in PCs-Mito-GFP mice after receiving the training with the simulating long-term pilot flight concentrating increased significantly. The precise reconstruction of neuronal mitochondrial networks is extremely laborious, the present 4-step workflow powered by artificial intelligence and virtual reality reconstruction could successfully address these challenges.

Nerve growth factor against PTSD symptoms: Preventing the impaired hippocampal cytoarchitectures.

Although exogenous nerve growth factor (NGF) demonstrated great potential for post-traumatic stress disorder (PTSD) treatment, its therapeutic effect and underlying cytological mechanism were not fully elucidated so far. We employed a controlled, prospectively designed modified single prolonged stress mice model to investigate the role of exogenous NGF on the modified single prolonged stress induced PTSD-like symptoms and hippocampal cytoarchitecture impairment, as well as the potential neuronal signaling modulation. We discovered that the modified single prolonged stress-exposure induced significant PTSD-like symptoms as well as mildly impaired hippocampal Cornu Ammonis 1 (CA1) subregion cytoarchitecture, but not dentate gyrus neurogenesis, together with a gradual inhibition of TrkA-CREB-ERK signalings in hippocampal CA1 subregion. NGF treatment dose-dependently ameliorated the modified single prolonged stress induced PTSD-like symptoms. NGF increased the cytoplasm/nucleus ratio and improved the neuronal plasticity, mainly via the TrkA-ERK-CREB pathway. Our study offered the translational evidence for the potential application of exogenous NGF for treating or early preventing PTSD after stress exposure.

Loss of Sfrp2 contributes to the neurological disorders related with morphine withdrawal via Wnt/ß-catenin signaling.

Morphine administration is a medical problem characterized by compulsive opioid use that causes terrible negative consequences. The exact mechanisms of morphine-induced dependence and morphine withdrawal symptoms remain unclear. Recent studies have revealed that the upregulation of Wnt/ß-catenin signaling plays important roles in morphine exposure and morphine withdrawal. Secreted frizzled-related protein 2 (Sfrp2) can prevent the activation of Wnt/ß-catenin signaling by competing with the Frizzled receptor for Wnt ligands. We conducted this study aimed to evaluate the effect of iatrogenic trauma induced by stereotactic surgery and the protective effect of stereotaxic Sfrp2 injection on morphine withdrawal symptoms in Male Sprague Dawley (SD) rats. Many techniques including western blot analysis and immunoprecipitation were used. Anxiety-related behaviors, morphine withdrawal syndrome, and dendritic spines were also examined in male SD rats after morphine treatment and stereotaxic injection of Sfrp2. Western blot results suggested that Wnt signaling was activated in the nucleus accumbens of SD rats suffering from morphine withdrawal and that Sfrp2 attenuated the overexpression of Wnt signaling. Similarly, the withdrawal-like symptoms of morphine dependent rats were abrogated by intracerebral Sfrp2 injection. The iatrogenic trauma induced by stereotactic surgery showed no influence on the Wnt signaling and withdrawal-like symptoms. Moreover, the results of Golgi-cox staining and DiI staining indicated that the damage on proximal spine density caused by morphine treatment was restored by intracerebral Sfrp2 injection. Together, the data presented here indicated that Sfrp2 abrogated the neurological disorders and loss of proximal spine related with morphine withdrawal via Wnt/ß-catenin signaling.

Pretreatment with Sodium Phenylbutyrate Alleviates Cerebral Ischemia/Reperfusion Injury by Upregulating DJ-1 Protein.

Oxidative stress and mitochondrial dysfunction play critical roles in ischemia/reperfusion (I/R) injury. DJ-1 is an endogenous antioxidant that attenuates oxidative stress and maintains mitochondrial function, likely acting as a protector of I/R injury. In the present study, we explored the protective effect of a possible DJ-1 agonist, sodium phenylbutyrate (SPB), against I/R injury by protecting mitochondrial dysfunction via the upregulation of DJ-1 protein. Pretreatment with SPB upregulated the DJ-1 protein level and rescued the I/R injury-induced DJ-1 decrease about 50% both in vivo and in vitro. SPB also improved cellular viability and mitochondrial function and alleviated neuronal apoptosis both in cell and animal models; these effects of SPB were abolished by DJ-1 knockdown with siRNA. Furthermore, SPB improved the survival rate about 20% and neurological functions, as well as reduced about 50% of the infarct volume and brain edema, of middle cerebral artery occlusion mice 23 h after reperfusion. Therefore, our findings demonstrate that preconditioning of SPB possesses a neuroprotective effect against cerebral I/R injury by protecting mitochondrial function dependent on the DJ-1 upregulation, suggesting that DJ-1 is a potential therapeutic target for clinical ischemic stroke.

Overexpression of NDRG2 can inhibit neuroblastoma cell proliferation through negative regulation by CYR61.

Several recent studies have showed that the n-myc downstream regulated gene 2 (NDRG2) is a new tumor suppressor gene, and that it plays an important role in tumor suppression in several cancers or cancer cell lines. However, few studies focused on its function in neuroblastoma cells. In the present investigation, we demonstrated that NDRG2 overexpression inhibited their proliferation. Using a cDNA microarray, we found that overexpression of NDRG2 inhibited the expression of cysteine-rich protein 61 (CYR61), a proliferation related gene. From our research, CYR61 may partially hinder NDRG2-mediated inhibition of cell proliferation. Overexpression of NDRG2 resulted in accumulation of cells in the G1 phase, which was accompanied by upregulation of p21 and p27 and downregulation of CDK4 and cyclin D1. Taken together, these data indicate that NDRG2 inhibits the proliferation of neuroblastoma cells partially through suppression of CYR61. Our findings offer novel insights into the physiological roles of NDRG2 in neuroblastoma cell proliferation, and NDRG2 may prove to be effective candidate for the treatment of children with neuroblastoma.

The effect of adenovirus-conjugated NDRG2 on p53-mediated apoptosis of hepatocarcinoma cells through attenuation of nucleotide excision repair capacity.

NDRG2 mRNA and protein levels can be upregulated in a p53-dependent manner. NDRG2 enhances p53-mediated apoptosis, whereas overexpression of NDRG2 suppresses tumor cell growth, regardless of whether p53 is mutated. However, the complicated mechanism by which NDRG2 suppresses tumor cell growth and enhances apoptosis mediated by p53 is not fully understood. Here, we demonstrated that Ad-NDRG2 enhanced the apoptosis of HepG2 cells (wild-type p53). Additionally, Ad-NDRG2 combined with rAd-p53 enhanced the apoptosis of Huh7 cells (mutant p53) after chemotherapy, and the expression of the ERCC6 gene (Cockayne syndrome group B protein gene) was suppressed in this process. Ad-NDRG2 combined with rAd-p53 induced the apoptosis of tumor cells (HepG2 and Huh7 cells); however, apoptosis was attenuated after transfection with ERCC6. Our results indicate that Ad-NDRG2 enhances the p53-mediated apoptosis of hepatocarcinoma cells (HepG2 and Huh7) by attenuating the nucleotide excision repair capacity (i.e., by downregulating ERCC6), and ERCC6 is a NDRG2-inducible target gene that is involved in the p53-mediated apoptosis pathway.

Estrogen regulates the expression of Ndrg2 in astrocytes.

N-myc downstream-regulated gene 2 (Ndrg2) is a newly identified molecule that is mainly expressed in astrocytes within the central nervous system (CNS) and is involved in the proliferation and activation of astrocytes. 17ß-estradiol (E2) is one of the most important circulating hormones, and in the CNS, astrocytes are a target and potential mediator of the action of E2. Our most recent study found that DPN, an estrogen receptor (ER) ß-specific agonist, activated the Ndrg2 promoter and elevated endogenous NDRG2 protein expression in MCF7, HSG and T-47D cells. However, whether E2 regulates Ndrg2 expression in astrocytes remains unknown. Here, we conducted both in vivo and in vitro experiments and found that ERß co-localized with NDRG2 in astrocytes. Furthermore, in primary cultured astrocytes, we demonstrated that E2 up-regulated Ndrg2 mRNA and protein expression in a dose- and time-dependent manner and that the ERß agonist DPN but not the ERα agonist PPT up-regulated Ndrg2 expression. In vivo, we found that in the hippocampus of adult ovariectomized (OVX) female mice, Ndrg2 mRNA and protein expression were significantly decreased compared with those in normal adult female mice. After the OVX mice received continuous subcutaneous injections of 50µg/kg E2, 100µg/kg E2 or the ERß agonist DPN for 10 days, the Ndrg2 expression significantly increased compared with that of the OVX mice. Our results indicate that E2 may affect astrocytes by regulating Ndrg2 expression.

Effect of transarterial pulsed perfusion with heated saline on tumor vascular permeability in a rabbit VX2 liver tumor model.

PURPOSE: To evaluate the effect of transarterial pulsed perfusion with 60 °C saline on vascular permeability of tumor tissue, as well as its hepatic and renal toxicity, in a rabbit VX2 liver model. MATERIALS AND METHODS: VX2 carcinomas were grown in rabbit livers, forty male New Zealand white tumor-bearing rabbits were randomly divided into four groups, followed by transarterial perfusion with 37 °C saline 60 ml (n=10) (control 1 group), transarterial pulsed perfusion with 37 °C saline 60 ml (n=10) (control 2 group), transarterial continuous perfusion with 60 °C saline 60 ml (n=10) (TCP group), transarterial pulsed perfusion with 60 °C saline 60 ml (n=10) (TPP group), the duration of time for tumor tissues in the range 43-45 °C of the treated groups was measured with needle thermometer during perfusion. Vascular permeability was assessed using the extravasation of Evans blue (EB) dye in the tumor or normal liver tissues of the four groups separately, the tumor or normal liver tissues of the four groups were estimated by histopathologic examination, and hepatic and renal toxicity was evaluated by means of blood biochemical analysis. The vascular endothelial cells in the tumor were observed by transmission electron microscopy (TEM). RESULTS: The duration of time for tumor tissues in the range 43-45 °C of TPP group showed significantly longer than that of TCP group (12.3±3.3 min vs. 5.7±2.5 min) (P<0.01). After perfusion, the EB content of tumor tissue in TPP group showed significantly higher than that in TCP group (15.21±0.94 µg/100 mg vs. 10.71±0.84 µg/100 mg) (P<0.01), and also showed significantly higher than that in the two control group (3.42±0.87 µg/100 mg, 3.57±0.64 µg/100 mg) (P<0.01). Blood chemical analysis indicating there was an increase (P<0.05) in the serum ALT, AST levels in the two heated perfusion groups at 1, 2, 4, 8 h after infusion when compared to that in the two control group, but there was no significant difference in the serum ALT, AST levels among the four groups at 24 h after perfusion (P>0.05), and there was no significant difference in the serum BUN, Cr levels among the four groups at 1, 2, 4, 8, 24 h after perfusion. Observed by hematoxylin and eosin staining, there were no obvious signs of tissue destruction in liver tissue and tumor tissue. TEM indicating the endothelial cell gap was broadened and the endothelial cells' microvillus was decreased after heated perfusion. CONCLUSIONS: The vascular permeability of the rabbit VX2 tumor was significantly increased after transarterial pulsed perfusion with 60°C saline without significant increase in hepatic and renal toxicity.